CHAPTER 5

Vascu l

Roles

Julio Sch

Contents

Trypanosoma cruzi invasion of cardiovascular

References 120Advances in Parasitology, Volume 76 # 2011 Elsevier Ltd.ISSN 0065-308X, DOI: 10.1016/B978-0-12-385895-5.00005-0 All rights reserved.

Instituto de Biofsica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS, Laboratorio deImunologia Molecular, Cidade Universitaria Rio de Janeiro, Rio de Janeiro, Brazilcells 1085.2.3. Interstitial oedema induced by

trypomastigotes: Role of the kinin system 1125.2.4. ACE is a negative modulator of TH1 induction

by kinin danger signals released in peripheralsites of infection 114

5.2.5. DCs activated by kinins induceimmunoprotective type-1 effector T cells inmice systemically infected by Trypanosomacruzi 117

5.3. Future Directions 118Acknowledgements 1205.2.1. Mechanisms underlying infection-associatedvasculopathy 105

5.2.2. Bradykinin receptors: A gate of entry forC

A Brief Overview on the Immunopathogenesis ofhagas Disease 1035.1. In5.2.troduction 103arfstein and Daniele AndradePathwaysKinin

of Endothelin andChaga

lopathy in Experimentas Disease: PathogenicInfection-Associated101

Abstract Acting at the interface between microcirculation and immunity,Trypanosoma cruzi induces modifications in peripheral tissues

nd

lly

generated by cruzipain. Further downstream, kinins stimulate lymph

ABBREVIATION

ACE angBKRs braCCM chrcruzipain maCTLs cytDCs deET endHCP hamHK higHUVECs huKKS kalPRR pa

102 Julio Scharfstein and Daniele Andradenode dendritic cells via G-protein-coupled BK2R, thus converting

these specialized antigen-presenting cells into TH1 inducers. Tightly

regulated by angiotensin-converting enzyme, the intact kinins (BK2R

agonists) may be processed by carboxypeptidase M/N, generating

[des-Arg]-kinins, which activates BK1R, a subtype of GPCR that is

upregulated by cardiovascular cells during inflammation. Ongoing

studies may clarify if discrepancies between proinflammatory

phenotypes of T. cruzi strains may be ascribed, at least in part, to

variable expression of TLR2 ligands and cruzipain isoforms.

S

iotensin-converting enzymedykinin receptorsonic chagasic myocardiopathyjor cysteine protease of T. cruziototoxic CD8 T cellsndritic cellsothelinster cheek pouch

h molecular weight kininogenman umbilical vein endothelial cellslikreinkinin systemttern-recognition receptorsbradykinin receptors, respectively, engaged by tGPI (TLR2 ligand) a

kinin peptides (bradykinin B2 receptors (BK2R) ligands) proteolyticainterstitial oedema in peripheral sites of infection through synergis-

tic activation of toll-like 2 receptors (TLR2) and G-protein-coupledinflammation revealed that tissue culture trypomastigotes elicitgenic inflammation to immunity. Analyses of the dynamics ofsystem emerged as a proteolytic mechanism that links oedemato-by infected cardiovascular cells. Paralleling these studies, the kininmyocardial fibrosis is worsened as result of endothelin upregulationpathogenesis came from research in animal models showing thator parasite-derived eicosanoids (thromboxane A2). Initial insight into(cruzipain), surface glycoproteins of the trans-sialidase family and/vasculopathy to the proinflammatory activity of a small subset of

T. cruzi molecules, namely GPI-linked mucins, cysteine proteasesthis chapter, we will review evidence linking infection-associatedwhich translate into mutual benefits to host/parasite balance. In

the clinical pleiomorphism of Chagas disease might result from the

entaingincotsisgrese

Role of Kinins and Endothelins in Chagasic Vasculopathy 103nism drivinshowing pCCM was called into question by independent studiesnce of traces of DNA or T. cruzi antigens in heart tissuesthe hypothe

definitively resolved (Teixeira et al., 2011), in the mid 90sthat autoimmunity was the primary pathogenic mecha-were the prtroversy is nipal effectors of cardiac inflammation. Although the con-

the underly premise being that self-reactive (anti-heart) lymphocytes

CCM was t tively classified in textbooks as an autoimmune disease,interplay between the genetically diversified T. cruzi species and thevariable genetic make-up of the human host (Andrade, 1999; Macedoand Pena, 1998; Vago et al., 2000; Venegas et al., 2009). In spite of earlyevidences that T. cruzi diversification resulted from clonal evolution ofancestor lineages (Tibayrenc et al., 1986), it was recently recognized thatgenetic exchange has also produced hybrid ancestor lineages that furthercontribute to the variability of currently circulating strains (De Freitaset al., 2006; Sturm and Campbell, 2009; Westenberger et al., 2005). Follow-ing recommendations made by an expert panel (Zingales et al., 2009), itwas recently proposed that the isolates/strains of T. cruzi should beclassified in six ancestor lineages, designated as TcI to TcVI. In the presentchapter, we will discuss in general terms the impact of genetic diversifica-tion of T. cruzi on oedematogenic inflammation.

5.2. A BRIEF OVERVIEWON THE IMMUNOPATHOGENESIS OFCHAGAS DISEASE

In the early 1970s, pathologists were intrigued with the observation thatT. cruzi pseudocysts were rarely detected in myocardial specimens fromchronic chagasic patients, in spite of the presence of extensive inflamma-tory infiltrates and tissue fibrosis (Andrade et al., 1994). For several years,TCTs tissue culture trypomastigotestGPIm trypomastigote-derived glycosylphosphatidylinositol-

anchored mucin-like glycoproteins from T. cruziTLR2 toll-like 2 receptorsTS trans-sialidaseTXA2 thromboxane A(2)

5.1. INTRODUCTION

After decades of systematic investigations, the concept that low-gradetissue parasitism is the primary mechanism leading to chronic chagasicmyocardiopathy (CCM) is firmly established. Although this hypothesispredicts that Trypanosoma cruzi antigens and/or proinflammatorymolecules play a central role in CCM, there is growing awareness that

104 Julio Scharfstein and Daniele Andradeof chagasic patients (Benvenuti et al., 2008; Higuchi et al., 1997; Palominoet al., 2000). The notion that T. cruzi organisms are directly involved indisease outcome was further substantiated by evidences that chronicpatients displaying the cardiac forms of Chagas disease bear parasiteDNA in the heart, but not in oesophageal tissues, whereas, reciprocally,the patients that exclusively develop gastrointestinal abnormalities showthe presence of parasite DNA in oesophagus tissues, but not in the heart(Jones et al., 1993; Vago et al., 1996). While these human pathologicalstudies were in progress, research in animal models showed that immunecontrol of T. cruzi infection depends on the integration between humoraland the cellular (innate and adaptive) branch of anti-parasite immunity(Tarleton et al., 1994). Concerning the T cell-dependent branch of immu-nity, the analysis of the epitopes recognized by class I MHC-restrictedeffector CD8 T cells identified the trans-sialidase (TS) family of antigensas dominant targets in both humans and mice (Garg and Tarleton, 2002;Tzelepis et al., 2008; Wizel et al., 1997). Given the extensive polymorphismobserved in TS antigens, these results initially suggested that the immunesystem is able to efficiently reduce parasite tissue burden in the acutephase by focusing the effector CD8 T cell responses on a limited range ofdominant TS peptides, consequently bringing the intracellular level ofinfection to limits that are compatible with host survival (Wizel et al.,1997). Adding complexity to this picture, subsequent studies revealedthat the hierarchy of immunodominant TS epitopes recognized by effec-tor CD8 T cells varies from one T. cruzi strain to another (Martin et al.,2006; Tzelepis et al., 2008). Since naturally infected hosts are often exposedto multiple T. cruzi clones, Martin et al. (2006) hypothesized that stochas-tic expression of variant TS epitopes by intracellular amastigotes and/ortrypomastigotes may allow for parasite escape from the immuneresponse, thus providing a driving force for the evolutionary diversifica-tion of TS family genes. More recently, Rosenberg et al. (2010) challengedthe concept that resistance to infection is critically dependent on thegeneration of TS-specific effector CD8 T cells recognizing dominantTS-encoded epitopes. In an elegant study, they showed that mice previ-ously tolerized by high-dose injections of dominant TS peptides wereresistant to an acute challenge, implying that the mice are able to effec-tively combat T. cruzi by generating effector T cells that recognize sub-dominant epitope specificities, not necessarily encoded by TS familymembers.

Despite the wealth of information emerging from immunologicalstudies in animal models, it is not obvious why a small proportion ofT. cruzi organisms subvert clearance by effector CD8 T cells. Initialstudies suggested that endogenous suppressive factors generated in theinflamed muscle tissue may limit the efficacy of cytotoxicity mediated byCD8 T cells (Leavey and Tarleton, 2003). Additional studies suggested

profiles of intracardiac T cells isolated from cardiac versus indeterminate

Role of Kinins and Endothelins in Chagasic Vasculopathy 105chagasic patients (Fonseca et al., 2007), immunologists relied on lympho-cytes isolated from peripheral blood to analyze systematically the profileof antigen-experienced T cells from chronic patients. Using epimastigoteantigens, Gomes et al. (2003) were able to categorize the immune respon-sive profile of chagasic patients based on IFN-g production by CD4 Tcells. In their study, the frequency of type-1 responders was significantlyhigher among cardiac patients, whereas low type-1 responders predomi-nated in patients with indeterminate disease. Interestingly, the low IFN-gproduction observed in indeterminate patients was inversely correlatedwith high frequencies of IL-10-producing monocytes (Gomes et al., 2003).More recently, Souza et al. (2007) reported that patients with the indeter-minate form of Chagas disease display a higher ratio of IL-10 over TNF-a-producing monocytes. Along similar lines, Araujo et al. (2007) found thatindeterminate patients display a higher percentage of CD4CD25 T cellsexpressing FOXP3 and IL-10. Adding substance to these in vitro studies,Costa et al. (2009) reported that patients exhibiting polymorphism of anIL-10 promoter gene associated to lower expression levels of the IL-10regulatory cytokine had a higher frequency of heart disease. Collectively,the studies with peripheral blood cells suggest that patients with asymp-tomatic/attenuated heart disease may rely on IL-10 producing macro-phages and/or regulatory T cells to limit the collateral damage which isotherwise inflicted by intracardiac TH1-type effector cells.

5.2.1. Mechanisms underlying infection-associatedvasculopathy

In the early 1990s, experts in vascular pathology advanced the proposi-tion that infection-associated vasculopathy could induce cumulativedamage in the chronically parasitized myocardium, perhaps renderingthe heart tissues more vulnerable to antigen-induced immunopathology(Morris et al., 1990; Rossi, 1990). Years later, refined histochemical studiesrevealed a derangement of the microcirculation and abnormal interstitialmatrix patterns in the heart sections of CCM patients (Higuchi et al.,that differentiation of effector cytototoxic CD8 T cells (CTLs; Albaredaet al., 2006; Grisotto et al., 2001) may be hampered as a result of dysfunc-tions occurring in the memory T cell compartment of TS-specific T cells.

Although the nature of the mechanisms underlying immune subver-sion is still uncertain, there were mounting evidences linking thelow-grade myocardial parasitism to the presence of inflammatory infil-trates enriched in TNF-a-producing CD8 T cells in the heart of patientswith chronic myocardiopathy (Reis et al., 1993) or in experimentallyinfected mice (Tarleton, 2003; Zhang and Tarleton, 1996). Given the tech-nical obstacles to compare the antigen specificity and immune response

1999). Paralleling these human studies, investigations carried out in the

106 Julio Scharfstein and Daniele Andrademouse model of Chagas disease suggested that endothelin-1 (ET-1) couldcontribute to infection-associated vasculopathy (Tanowitz et al. 1999).Constituted by a family of three peptides (ET-1, ET-2 and ET-3) of 21amino acids encoded by distinct genes, endothelins are expressed byendothelial cells, cardiac myocytes and cardiac fibroblasts (Goto, 2001;Kedzierski and Yanagisawa, 2001). Synthesized as prepro-endothelin,these precursor proteins are cleaved by endothelin-converting enzymesforming big-endothelin, which upon further processing yields peptidesthat activate cells via G-protein-coupled receptors (GPCRs; for review, seeDhaun et al., 2007). Endothelin is involved in a host of physiologicalprocesses via the activation of two GPCR subtypes, ETA and ETB.Endowed with powerful vasoconstrictor function, ET-1 is also able tomodulate the expression of leukocyte adhesion molecules on endothelialcells and on fibroblast-like synovial cells (Schwarting et al., 1996), inducesplasma exudation and oedema formation (Filep et al., 1993; Sampaio et al.,2000), stimulates cytokine production (Sampaio et al., 2000; Speciale et al.,1998) and regulates neutrophil adhesion and migration (Sampaio et al.,2000; Zouki et al., 1999).

After reporting that the plasma levels of ET-1 are increased both inchagasic patients and in mice (Petkova et al., 2000; Salomone et al., 2001),these authors documented that ET-1 (i) expression is upregulated inT. cruzi-infected cardiovascular cells (endothelial cells and cardiac myo-cytes; Petkova et al., 2000) and (ii) induces vasospasm in T. cruzi-infectedmice, hence contributing to the development of myocardial ischaemia andmyonecrosis (Tanowitz et al., 2005). These authors demonstrated thatcardiac remodelling was ameliorated in T. cruzi-infected mice in whichthe ET-1 gene was deleted exclusively from cardiac myocytes (Tanowitzet al., 2005). Based on these findings, the authors advanced the proposi-tion that ETR antagonists might be considered in adjunctive therapy ofchagasic heart disease (Mukherjee et al., 2004; Tanowitz et al., 2005).

Further insight on infection-associated vasculopathy emerged fromstudies of the pathogenic roles of T. cruzi prostanoids (Ashton et al.,2007). These authors focused their attention on thromboxane (TXA2),after pondering that the multiple vascular sequelae associated withT. cruzi infection could relate to the upregulated function of this eicosa-noid, for example, denudation of the endothelium (leading to increasedvascular permeability) and increased expression of leukocyte adhesionmolecules on the endothelium. In addition, TXA2 promotes proliferationand migration of smooth muscle cells, thus contributing to neointimaformation (Ashton et al., 2007). Research focusing on TXA2 could alsoshed light on dysfunctions in haemostasis, since this eicosanoid promotesplatelet activation/aggregation and degranulation. Importantly, Ashton

Role of Kinins and Endothelins in Chagasic Vasculopathy 107et al. (2007) reported that mice deficient in the thromboxane receptor (TP)bear a highly susceptible phenotype, characterized by increased mortal-ity, cardiac pathology and higher tissue parasitism. After showing thatTXA2 is the predominant eicosanoid lipid produced in the blood ofchagasic mice, the authors demonstrated that up to 90% of the circulatinglevels of TXA2 were of parasite origin, rather than from the host. Interest-ingly, the levels of TXA2 produced by amastigotes are significantly higherthan those of trypomastigotes or epimastigotes. Clues to understand thepotential significance of these findings emerged from analysis of theoutcome of infection in cultures of endothelial cells derived fromwild-type versus TP-deficient mice; the authors noted that the infectionindex was markedly increased in the mutant mice. Based on these obser-vations, the authors proposed that TP, most likely triggered by amasti-gote-derived TXA2, may fine-tune the rate of intracellular parasitegrowth, preventing dysregulated expansion of the intracellular load ofparasites within endothelial cells. Extending these studies to the in vivosettings, Ashton et al. (2007) observed that T. cruzi-TP-null mice displayedan increased mortality, parasite tissue load and cardiac pathology.Infections employing bone marrow chimeric mice argued against thepossibility that TP deficiency in immune cells might account for thesusceptible phenotype of TP-null mice. These results, combined withthe culture studies performed with endothelial cells, suggest that theTXA2/TP axis may limit parasite infectivity in somatic cells, throughmechanisms that remain unclear.

Another area of research linking T. cruzi activity to endothelium injuryemerged from studies on the pathogenic role of TS. Progress in this fieldstarted with the observation that endothelial cells and cardiomyocytessuffered de-sialylation upon treatment with T. cruzi neuraminidase(Libby et al., 1986), the latter being described as a TS (Previato et al.,1985; Zingales et al., 1987). More recently, Dias et al. (2008) used catalyti-cally inactive recombinant TS to characterize in further details themolecular basis of TS binding to endothelial cells. Their data showedthat TS binds to endothelial cell surface a2,3-linked sialic acid residuesthrough a lectin-binding site. Functional analysis of the outcome of thelectin site of TS with the endothelium revealed that the interaction (i) ledto the activation of NF-kB, (ii) increased expression of adhesion moleculesand (iii) reduced apoptosis upon endothelial cell exposure to growthfactor deprivation (Dias et al., 2008). Focusing a novel aspect of TSresearch, that is, the molecular mechanism involved in endotheliumtransmigration and tissue tropism, Tonelli et al. (2010) postulated thattrypomastigotes might interact with microvascular beds through thebinding of a conserved peptide motif of TS shared by several membersof the polymorphic T. cruzi family. The presence of circulating antibodiesto TS (Duthie et al., 2005) may also account for the infection-associated

from an internal moiety of high (HK) or low (LK) molecular weightkininogens (Bhoola et al., 1992). Although kinins are traditionally viewed

108 Julio Scharfstein and Daniele Andradeas classical mediators of acute inflammation (e.g. inducers of oedemaformation, vasodilation and pain sensations), it is now well establishedthat these short-lived peptide hormones may modulate the microcircula-tion homeostasis (Bhoola et al., 1992; Schmaier, 2004). As discussed laterin this chapter, knowledge emerging from studies of the KKS role inimmunity has linked the role of kinins to the IL-12-dependent cytokinemicroangiopathy described in chagasic patients (Higuchi et al., 1999) andexperimentally infected mice (Andrade et al., 1994). For example, endo-thelium decorated with TS molecules that are shed by trypomastigotesmight be injured as result of antibody-mediated cellular cytotoxicity,reminiscent of the bystander mechanism of host cell death originallyenvisaged by Ribeiro Dos Santos and Hudson (1981).

5.2.2. Bradykinin receptors: A gate of entry forTrypanosoma cruzi invasion of cardiovascular cells

Given the low level of intracellular parasitism observed in the myocar-dium of chronic patients, we may predict that the interstitial spaces of theheart are only sporadically exposed to intracellular T. cruzi released frompseudocysts, that is, the membrane-containing structures harbouringparasites at the final stages of their intracellular life cycle. Once releasedfrom pseudocysts, the trypomastigoteswhich for operationalreasons will be henceforth designated as tissue culture trypomastigotes(TCTs)rapidly move away from the primary foci of infection, seekingfor a safer environment (i.e. non-inflamed) to efficiently propagate theinfection. As previously suggested (Scharfstein and Morrot, 1999), it ispossible that premature killing of parasitized target cells by amastigote-specific MHC Class I restricted CTLs may lead to the release of amasti-gotes to the heart interstitium. Devoid of a moving flagellum, the amas-tigotes tend to cluster in the surroundings of the primary infection foci,perhaps accounting for most, if not all, of the parasite antigens detected inheart specimens of CCM patients (Higuchi et al., 1999). As reviewedbelow, the immunohistochemical identification of cruzipain depots inthe myocardium of CCM patients (Morrot et al., 1997) suggested thatthis major T. cruzi antigen could play a role in immunopathology(Scharfstein, 2010). While these immunological studies were in progress,Scharfstein and co-workers realized that enzymatically active cruzipainmay fuel inflammation through the activation of the kallikreinkininsystem (KKS; Del Nery et al., 1997; Lima et al., 2002).

The term kinin refers to a small group of vasoactive metabolitesrelated to the bradykinin (BK), a nonapeptide proteolytically released

circuitry that shapes T-cell development (Aliberti et al., 2003; Monteiro

Role of Kinins and Endothelins in Chagasic Vasculopathy 109et al., 2006, 2007, 2009).Due to their short life (half-life of

110 Julio Scharfstein and Daniele Andradefrom Staphylococcus aureus (Imamura et al., 2005) and streptopain fromStreptococcus pyogenes (Herwald et al., 1996).

The first clues indicating that T. cruzi activated the kinin system cameas a result of the studies by Del Nery et al. (1997) who analysed thesubstrate specificity properties of the major cysteine protease of T. cruzi(cruzipain). Classified as member of clan A of the C1 peptidase family(Cazzulo et al., 1989), cruzipain is a well-characterized therapeutictarget in Chagas disease (Doyle et al., 2007). Substrate specificity studiesperformed with intramolecularly quenched fluorogenic peptides span-ning the N- and C-terminal flanking sites of the lysyl-BK sequence, DelNery et al. (1997) revealed that cruzipain resembles tissue kallikrein, thatis, both enzymes are able to cleave HK, releasing the internal lysyl-BKmoiety. Initially, the discovery that cruzipain is a kininogenase seemedparadoxical because kininogens are members of the cystatin family ofcysteine protease inhibitors, hence rely on cystatin-like domains topotently inactivate papain-like enzymes, including cruzipain (Stokaet al., 1995). Noteworthy, however, the studies performed by Del Neryet al. (1997) revealed that purified cruzipain was able to release bioactivekinins from soluble forms of HK, but unlike tissue kallikrein, the reactionoccurred at slow rates. The conundrum was settled after considering thatHK binds to endothelial cells through two distinct domains: (i) a domain(D3) that overlaps with the cystatin domain (Herwald et al., 1995) and (ii)a histidine-rich positively chargedmotif (D5H) localized at the C-terminalend of the BK (D4) sequence, which binds to negatively chargedsulphated proteoglycans, such as heparan or chondroitin sulphates(Renne et al., 2000; Renne and Muller-Esterl, 2001). Based on this infor-mation, Lima et al. (2002) hypothesized that the spatial orientation of cell-boundHK docked to heparan sulphate proteoglycans was not suitable forcruzipain binding and inactivation by the cystatin-like inhibitory domain.Indeed, model studies performed with cruzipain and HK in the test tubeoffered circumstantial support to this hypothesis: the addition of heparansulphate (tested at optimal concentrations) drastically reduced the cyste-ine inhibitory activity of soluble HK on cruzipain, while reciprocallyincreasing the catalytic efficiency (sixfold) of the parasite protease. Con-sistent with these findings, the addition of heparan sulphate increased theefficiency of the kinin-releasing activity of cruzipain (albeit only at rela-tively narrow concentration range) and resulted in the formation of mul-tiple HK breakdown products. Combined, these biochemical studiessuggested that the substrate specificity of the parasite protease was redir-ected as result of reciprocal interactions between sulphated proteoglycanswith the substrate (HK) and protease (cruzipain) molecules, henceincreasing the efficiency of the kinin release reaction (Lima et al., 2002).

While these biochemical studies were in progress, Scharfstein et al.(2000) demonstrated that living TCTs (Dm28c) rely on the kinin-releasing

Role of Kinins and Endothelins in Chagasic Vasculopathy 111activity of cruzipain to infect cells that overexpress BK2Rs, such as humanumbilical vein endothelial cells (HUVECs) or CHO-transfected cell linesoverexpressing BK2R. After showing that TCTs induce strong [Ca

2]itransients via the cruzipain/BK2R pathway, the authors suggested thatparasite uptake involved the [Ca2]i/lysosomal pathway originallydescribed by Tardieux et al. (1992). Evidence linking the processing ofkininogens to cruzipain-dependent generation of the BK2R agonist wasobtained in invasion assays performed in the presence of exogenous HK.These studies showed that parasite uptake by CHO-BK2R was enhancedupon addition of purified HK or, alternatively, by addition of physiologi-cal concentration of BK (i.e. the BK2R agonist) into the serum-freemedium. Further, mAbs directed to kininogens blocked invasion onCHO-BK2R but did not interfere with the baseline levels of infection ofCHO mock, further suggesting that cell-bound kininogens serve asprecursors for the BK2R agonist(s) released by cruzipain (Scharfsteinet al., 2000). Another interesting revelation of this study was the evidencethat ACE/kininase II, a metallopeptidase that is strongly upregulated inHUVECs, limits the ability of the parasite to invade this particular celltype via the BK2R pathway.

In view of the technical obstacles to ablate the multiples cruzipaingenes, invasion assays were carried out with active-site directed cysteineprotease inhibitors. Unexpectedly, the results revealed that membrane-permeable cruzipain inhibitors markedly reduced extent of parasite inva-sion via the BK2R pathway, while addition of soluble inhibitors such ascystatin C or E-64 did not interfere at all with parasite infectivity(Scharfstein et al., 2000). Given that trypomastigotes are poorly endocytic(De Souza, 1995) and that these flagellates accumulate cruzipain in theflagellar pocket (Murta et al., 1990; Souto-Padron et al., 1990), Scharfsteinand co-workers reasoned that the kinin-releasing reaction may occur inenclosed areas formed by juxtaposition of host cell and parasite plasmamembranes, perhaps equivalent to a synapse (Tyler et al., 2005).This mechanistic model predicts that the lysosomal-like cruzipain mole-cules might diffuse from the parasites flagellar pocket into thisintercellular space, being thus spared from physiological inactivation bysoluble forms of plasma protease inhibitors (e.g. cystatins, kininogens,a2-macroglobulin) present in extracellular body fluids. Although notdirectly demonstrated, this concept also implies that surface-boundkininogens, along with bradykinin receptors (BKRs), are activelyrecruited to such signalling centres (Scharfstein et al., 2000).

Although BK2R was the first GPCR with defined pharmacologicalspecificity to be implicated in the [Ca2]/lysosomal pathway of T. cruziinvasion (Andrews, 2000; Burleigh and Woolsey, 2002; Leite et al., 1998),in vitro studies subsequently showed that the inducible BK1R may serveas gateway for infective trypomastigotes (Todorov et al., 2003). In order to

tigotes activate the kinin system in vivo. Using mouse paw oedema as a

112 Julio Scharfstein and Daniele Andradereadout, studies in BK2R/ or BK1R

/ mice infected with trypomasti-gotes revealed that BK2R mediates the early-phase vascular responses(23 h), whereas the upregulated BK1R pathway accounts for the latephase (24 h) reaction. Noteworthy, the oedematogenic inflammation inwild-type mice was consistently mild (in BALB/c mice) or negligible (B6mice), except for animals purposefully deprived of ACE activity by sys-temic administration of captopril before parasite inoculation. Theseresults underscored the importance of ACE/kininase II as a modulatorof inflammatory oedema in mice infected subcutaneously (s.c.) withDm28c trypomastigotes.

Given the possibility that blood vessel injury by needle injection couldsynergize with parasite products to propel activation of the KKS,Monteiro et al. (2006) analysed the impact of topical application ofDm28c trypomastigotes in microcirculatory preparations of the hamstercheek pouch (HCP). The results from intravital microscopy studiesrevealed that the parasites induce amild BK2R-dependent plasma leakageresponse in the HCP, consistent with the mouse oedema studies. In bothmodels, the vascular reactions were potentiated by captopril andmitigated by Z11777, a highly specific irreversible inhibitor of cruzipainsimulate the settings of inflammation, the authors examined the outcomeof T. cruzi interaction with (i) HUVECs pre-activated, or not, withlipopolysaccharides (LPS) (TLR4 ligand) and (ii) neonatal cardiomyo-cytes, which spontaneously express BK1R. Assays performed in the pres-ence of BK1R antagonists or kininase I inhibitors revealed that parasiteuptake was markedly reduced. Noteworthy, measurements of intracellu-lar amastigotes several days after the onset of infection confirmed that theearly blockade of BK1R reduced parasite burden in endothelial cells orcardiomyocytes in a direct proportional to the number of penetratingparasites. Noteworthy, T. cruzi trypomastigotes infected cell typesoverexpressing the inducible BK1R in the absence of ACE inhibitors,suggesting that carboxypeptidase N/M-dependent generation of[des-Arg]-kinins (BK1R ligand) is prioritized over ACE-dependent degra-dation of the intact kinins (BK2R ligand). Another interesting aspect thatemerged from the studies of hostparasite interaction was the evidence ofcrosstalk between BK2R and BK1R (Todorov et al., 2003). As discussedfurther below, it is possible that Dm28c T. cruzimay take advantage of theubiquitous B1KR pathway to opportunistically invade cardiovascularcells in the inflamed heart tissues.

5.2.3. Interstitial oedema induced by trypomastigotes: Role ofthe kinin system

Todorov et al. (2003) were the first to demonstrate that Dm28c trypomas-

Role of Kinins and Endothelins in Chagasic Vasculopathy 113(Doyle et al., 2007). These results strongly suggested that the level ofbioactive kinins generated in peripheral sites of T. cruzi infection(steady-state conditions) depends on the balance between cruzipainand ACE.

In a crucial observation, Monteiro et al. (2006) observed that Dm28cepimastigotes did not elicit significant FITC-dextran leakage in captopril-treated HCP, despite the fact that these avirulent parasite stages expresshigh levels of cruzipain. These results suggested that expression of cru-zipain was necessary but insufficient for trypomastigotes to induceplasma leakage via the BK2R pathway. Consistent with this hypothesis,purified cruzipain (enzymatically active) failed to induce plasma leakagein the captopril-treated HCP superfusate. However, the combination ofcruzipain and purified HK to captopril-HCP led to a full-blown plasmaleakage via the BK2R pathway. Based on these findings, Monteiro andco-workers proposed that the rate-limiting step governing extent of kininrelease by cruzipain is the level of plasma-borne kininogens availablein peripheral sites of infection. As a corollary, the authors predicted that(i) in steady-state tissues (i.e. in the absence of a pre-established inflam-mation), the levels of kininogens in interstitial spaces are not sufficientlyhigh to propitiate appreciable proteolytic release of vasoactive kinins,either in tissues exposed to avirulent epimastigotes or to purifiedcruzipain, and (ii) trypomastigotes might be empowered with proinflam-matory molecules (absent in epimastigotes) which rapidly induce thediffusion of plasma-borne proteins (including kininogens) into the inter-stitial spaces. Efforts to identify this putative molecule converged to theglycophosphatidyl-linked mucin anchor of trypomastigotes (tGPI),originally characterized as a potent TLR2 ligand by Almeida andGazzinelli (2001). According to these workers, tGPI possesses an unsatu-rated fatty acid at the sn-2 position (TLR2 agonist) of the alkylacylglycerolmoiety, which is absent in the counterpart GPI anchors of epimastigotes.Consistent with a role for tGPI, Monteiro and co-workers demonstratedthat Dm28 trypomastigotes failed to elicit appreciable oedema both inTLR2/ and in neutrophil-depleted mice, irrespective of treatment withACE inhibitors. Moreover, assays performed in captopril-treated mice(wild-type, BK2R

/, TLR2/ and neutrophil-depleted) injected withthe combination of purified tGPI (TLR2 ligand) and cruzipain (enzymati-cally active) demonstrated that tGPI and cruzipain synergisticallyinduced footpad oedema via the TLR2/neutrophil/BK2R-dependentpathway, while ACE/kininase II has an anti-inflammatory role, since itinterferes with the transcellular crosstalk between TLR2 and BK2R.

It is well established that activated neutrophils are capable of inducingendothelial barrier disruption through a variety of mechanisms (DiStasiand Ley, 2009). Intravital microscopy observations in HCP suggested thatneutrophils play a role in the dynamics of oedematogenic inflammation

114 Julio Scharfstein and Daniele Andrade5.2.4. ACE is a negative modulator of TH1 induction by kinindanger signals released in peripheral sites of infection

DCs are a heterogeneous population of professional antigen-presentingcells (APCs) that are widely but sparsely distributed in peripheral tissuesand lymphoid organs (Shortman and Naik, 2007). Strategically positionedin T cell-rich areas of secondary lymphoid tissues, the resident DCs arespecialized in antigen presentation to CD4 and CD8 T cells. Insteady-state conditions, immature DCs contribute to the maintenance ofperipheral tolerance because these APCs display MHC-restricted antigenpeptides to virgin T cells in the absence of co-stimulatory molecules.However, during infection, immature DCs develop the competence toinitiate adaptive immunity after sensing the presence of inflammatorycues (danger signals) generated in peripheral sites of infection and/orin the lymphoid tissue environment (Sansonetti, 2006). Once drained bylymphatics, microbial antigens and proinflammatory molecules (includ-ing kinins) are transported to the DC-rich cortical areas of the lymphnode. After internalizing antigens via specialized scavenger receptors,the lymphoid-resident DCs may spread their antigen cargo to lym-phoid-resident DCs via release of exosomes and/or apoptotic bodyuptake (Sansonetti, 2006). While the antigens are processed and presentedin MHC-restricted manner in the surface of these specialized APCs,induced by Dm28c trypomastigotes (Monteiro et al., 2006). After notingthat the peak of plasma leakage was sligthly delayed in relation to leuko-cyte mobilization, Schmitz et al. (2009) studied the role of innate receptorsas the initiators of T. cruzi-elicited inflammation. First, they demonstratedthat resident macrophages stimulated in vitro by Dm28c trypomastigotesrobustly secreted neutrophil-attracting CXC chemokines (KC/MIP-2) inTLR2-dependentmanner.Next, theyverified that repertaxin (CXCR2antag-onist) blocked neutrophil-dependent influx of plasma proteins into theinterstitial spaces, thus reducing the initial influx of plasma-borne kinino-gens (cruzipain substrate) in peripheral sites of infection (Fig. 5.1). Com-bined, these studies suggested that TLR2/CXCR2/neutrophils control therate-limiting step (kininogendiffusion to interstitial spaces) of themicrovas-cular response which is required for over activation of the kinin system inperipheral sites of T. cruzi infection (Fig. 5.1). Once formed, the vasoactivekinins amplify oedematogenic inflammation initiated by TLR2/CXCR2/neutrophils through positive feedback cycles of endothelium BK2R activa-tion,which canbe furtherprolongedat expenseof activationof the inducibleBK1R pathway (Todorov et al., 2003). In summary, the flow of informationbetween innate immunity (TLR2-driven) and the proteolytic wave(cruzipain/BK2R-driven) of inflammation is modulated by the kinin-degrading activity of ACE/kininase II.

FIGURE 5.1 Mechanistic model depicting how the proinflammatory activities of kinins

and endothelins may converge to aggravate myocardial pathology in Chagas heart

disease. Lower side of panel, sparsely distributed the heart of chronically infected

patients, the heart cells containing pseudocysts sooner or later disrupt, releasing

numerous trypomastigotes to the interstitial spaces. Acting as typical microbial PAMPS,

tGPI-mucin (TLR2 ligands) shed by the TCTs are sensed by TLR2 constitutively expressed

by resident macrophages (left side of panel). Next, the activated macrophages secrete

neutrophil-attracting CXC chemokines (KC/MIP-2), which in turn bind to CXCR2

expressed by neutrophils/endothelium (upper left). Neutrophils activated by CXC

chemokines secrete vascular permeability factors which then disrupt the integrity of the

endothelium barrier. This allows for incipient leakage of plasma proteins, including

kininogens and ET-1 (present at high levels in the blood of patients with cardiac disease)

into peripheral sites of infection (upper side of panel). T. cruzi trypomastigotes process

kininogens associated to GAGs, liberating kinins via cruzipain (CZP). The biological

activity of the short-lived kinins (BK2R agonist) is mitigated by the kinin-degrading

activity of ACE/kininase II. The vigour of the inflammation steered by the TLR2/CXCR2/

neutrophil pathway may eventually overcome the regulatory constraints imparted by

ACE/kininase II. The build-up in the extravascular levels of vasoactive kinins leads to

overt activation of the kinin system, due to feedback loops of activation of endothelium

BK2R/BK1R. Further downstream, T. cruzi may then take advantage of the odedemato-

genic inflammation to invade cardiovascular cells through the cooperative activation of

Role of Kinins and Endothelins in Chagasic Vasculopathy 115

the antigen-bearing DCs concomitantly sense the presence of microbe-derived danger motifs through distinct pattern-recognition receptors(PRRs), such as TLRs or intracellular NOD2-like receptors (NLR; Kumaret al., 2011). In addition, DCs may sense the threat to tissue integrity viareceptors for endogenous proinflammatory mediators, such as ATP, uricacid (Sansonetti, 2006) and BK (Aliberti et al., 2003; Monteiro et al., 2007).Stabilized by cognate interactions with co-stimulatory molecules (CD80/86, CD40 and MHC), the prolonged encounters between antigen-bearingDCs and nave T cells are essential for TCR activation. During the courseof DC/T cell interaction, the mature APCs deliver polarizing cytokines.For example, IL-12p-70 is critically required for TH1 development.

In 2003, our group reported that exogenous lysyl-BK (LBK) potently

116 Julio Scharfstein and Daniele Andradeinduces the maturation (upregulation of IL-12 and co-stimulatory mole-cules) on wild-type CD11c DCs while failing to elicit such responses inBK2R

/DCs (Aliberti et al., 2003). In keeping with these in vitro observa-tions, studies in ovalbumin-immunized mice confirmed that exogenousLBK induced TH1 polarization via the BK2R/IL-12-dependent innate path-way. Subsequently, Monteiro et al. (2006) suggested that kinins released inperipheral sites of T. cruzi infection upregulated IL-12 production byCD11c DCs in the draining lymph node and steered TH1 developmentvia the BK2R pathway. Noteworthy, these effects were only observed ininfected mice pretreated with captopril, thus implying that ACE/kininaseII offsets the linkage between innate immunity (TLR2 dependent) and thedownstream proteolytic pathways that guide TH1 development via theBK2R/IL-12-dependent pathway (Monterio et al., 2006; reviewed byScharfstein et al., 2008). Analysis of T cell recall responses to parasiteantigens by lymphocytes isolated from draining lymph nodes revealedthat TH1 induction was compromised in TLR2

/ or neutrophil-depletedmice. Importantly, the deficient TH1 responses of TLR2

/ or neutrophil-depleted mice were fully restored by mixing purified HK to the sus-pension of living trypomastigotes shortly before footpad injection.In both cases, the HK-dependent rescuing of TH1 responses was nullified

BKRs and ETRs (Andrade et al., 2011). The interstitial oedema driven by kinins is further

intensified (top, right), increasing the levels of ET-1 in the interstitial spaces. Sustained

inflammation may also lead to upregulated expression of B1KR in the myocardium,

offering a window of opportunity for parasite invasion of cardiovascular cells. The

increase in intracellular parasite load translates as upregulated expression of endothe-

lins, which may then aggravate infection-associated vasculopathy and myocardial

fibrosis via ETRs. In addition, the upregulated expression of BK1R in the endothelium

lining may favour the recruitment of circulating anti-parasite IFN-g/TNF-a-producingCD4 T effector and CD8 T effectors to the heart parenchyma. For the sake ofsimplicity, the panel does not illustrate the impact of TLR2/B2R activation on DC

maturation and on TH1 development, at early stages of T. cruzi infection (Monteiro et al.,

2006, 2007).

Although the subcutaneous model of T. cruzi served as paradigm to

Role of Kinins and Endothelins in Chagasic Vasculopathy 117investigate the role of KKS in mechanisms linking inflammation toimmunity, the impact on host resistance could not be determined becausethese mice resisted acute challenge with Dm28c T. cruzi. Seeking for analternative model, Monteiro et al. (2007) compared the phenotypes ofBK2R

/ mice and BK2R/ mice in the classical intraperitoneal model

of acute infection. Strikingly, the BK2R/ mice displayed a highly sus-

ceptible phenotype, succumbing to acute T. cruzi challenge within30 days. Efforts to characterize the mechanisms underlying the immunedysfucntion of BK2R

/ mice failed to reveal profound defects in theintralymphoid (spleen) at early stages of infection: the frequencies ofantigen-specific IFN-g-producing CD8 T cells and CD4 T cells werefairly similar in wild-type and BK2R-deficient mice. However, there was asignificant drop in the frequency of intracardiac type-1 effector T cells inBK2R-deficientmice. Further, as the acute infection progressed in BK2R

/

mice, the immune deficiency was intensified, involving both the extra-lymphoid and lymphoid compartment. Intriguingly, the decayed TH1response of BK2R

/ was accompanied by a corresponding rise in IL-17-producing T cells (TH17). The premise that the deficient adaptive responseof BK2R

/mice was a secondary manifestation resulting from impairedby HOE-140 or by mixing purified HK with trypomastigotes pretreatedwith K11777 (irreversible cruzipain inhibitor). Collectively, these experi-ments supported the concept that plasma-borne kininogens diffusing ininterstitial spaces undergo proteolytic processing by cruzipain, liberatingendogenous signals (kinins) that subsequently convert BK2R

/ CD11c

DCs into inducers of TH1 polarization (Scharfstein et al., 2007). Furtherindications that the TLR2/BK2R axis bridges inflammation to innate/adaptive immunity emerged from studies in a mouse model of mucosalinflammation induced by the periodonto-bacterium P. gingivalis (Monteiroet al., 2009). Acting cooperatively, P. gingivalis LPS (TLR2 ligand) and gingi-pains (kinin-releasing proteases) induce mucosal inflammation and stimu-late antibacterial (fimbriae antigens) TH1/TH17 responses via the previouslydescribed trans-cellular TLR2/BK2R crosstalk. Notably, in contrast to theT. cruzi infection model, ACE inhibitors did not interfere with B2R-drivenstimulation of antibacterial TH1/TH17 responses in the P. gingivalis infectionmodel. Although not addressed experimentally, it is likely that the require-ment for ACE blockadewas superfluous in themodel of P. gingivalis-elicitedmucosal inflammation because gingipains are not sensitive to inhibition bythe cystatin-like domains of soluble kininogens.

5.2.5. DCs activated by kinins induce immunoprotective type-1effector T cells in mice systemically infected byTrypanosoma cruziBK2R/ DC maturation was confirmed by systemically injecting

showed that trypomastigotes pretreated with the irreversible cruzipaininhibitor (Z11777) failed to robustly activate wild-type DCs, thus suggest-

118 Julio Scharfstein and Daniele Andradeing that the BK2R agonist (DC maturation signal) is indeed released bycruzipain. Dm28c trypomastigotes induced the maturation of splenicCD11c DCs derived from TLR2/ and TLR4 mutant (C3H/HeJ) viaBK2R, thus precluding cooperative signalling between this GPCRs andeither PRRs. While not excluding the contribution of TLR9 (Bafica et al.,2006) or NOD2 (Silva et al., 2010) as potential sensors of T. cruzi, theseresults were consistent with the concept that kinin danger signals pro-teolytically released by trypomastigotes activate BK2R

/DCs, convertingthese APCs into inducers of type-1 immunity (Monteiro et al., 2007;Scharfstein et al., 2007). Since the spleen is continuously exposed to plasmaproteins, it is conceivable that Dm28 trypomastigotes might be faced withabundant levels of blood-borne kininogens bound to their docking sites (e.g., sulfate proteoglycans) present on cell surfaces and/or extracellularmatrixes. As a corollary, we may predict that the levels of kinin dangersignals proteolytically generated in the parasitized/inflamed splenicstroma may suffice to convert conventional CD11c DCs into TH1 indu-cers. As part of an initial effort to determine if some of these mechanisticprinciples are extended to the settings of human infection, Coelho dosSantos et al. (2010) have recently reported that ACE inhibitors converthuman monocytes into drivers of TH17-type responses against T. cruzi.

5.3. FUTURE DIRECTIONS

Focusing on the molecular pathways that govern host-parasite interac-tions at the interface between the microcirculation and immunity, in thischapter we have reviewed experimental findings indicating that infec-tion-associated vasculopathy may be linked to the proinflammatory activ-ities of a limited group of T. cruzi molecules. Special attention was givento discuss progress made in endothelin research, which culminated inthe discovery that myocardial fibrosis is aggravated as result of ET-1upregulation by T. cruzi-infected cardiomyocytes (Tanowitz et al., 2005).wild-type BK2R/ DCs (i.v.) into the susceptible BK2R

/ mice beforeinjecting the pathogen. Remarkably, this DC transfer manoeuvre renderedthe recipient BK2R

/ mice resistant to acute T. cruzi challenge andrestored their capability to generate protective IFN-g-producing CD4

CD44 and CD8 CD44 effector T cells, while conversely suppressingthe potentially detrimental TH17 (CD4

subset) anti-parasite responses.Using expression of IL-12 and co-stimulatory molecules (CD86, CD80,CD40) as readout for DCmaturation in vitro, Monteiro et al. (2007) furtherdemonstrated that Dm28c trypomastigotes potently activate BK2R

/

CD11cDCs (splenic origin) but not BK2R/DCs. Moreover, the authors

Role of Kinins and Endothelins in Chagasic Vasculopathy 119Adding further complexity to this picture, Andrade et al. (2011) haverecently reported that T. cruzi trypomastigotes (Dm28c strain) evokeedematogenic inflammation and invade cardiovascular cells throughmechanisms involving interdependent signaling of ETAR, ETBR andBK2R (Andrade et al., 2011). Based on these findings, the authors hypothe-sized that the trypomastigotes may take advantage of the accumulation ofplasma borne-proteins (including kininogens) and endothelins (ET-1) inextravascular tissues to infect cardiovascular cells more efficientlythrough the cooperative activation of ETRs/BKRs (Fig. 5.1). Future stud-ies may clarify if T. cruzi trypomastigotes may also exploit the inducibleBK1R to persist in the inflamed myocardium. This possibility comes tomind, in light of evidences emerging from research in diabetes andhypertension, showing that prooxidative signals generated by ET-1 andangiotensin II are able to upregulate B1KR expression in vascular smoothmuscle cells (Morand-Contant et al., 2010). In view of this interestingprecedent, we may predict that ET-1-driven induction of BK1R expressionin cardiovascular cells may offer a window of opportunity for parasiteinvasion of cardiovascular cells via the inducible kinin pathway (Fig. 5.1).Furthermore, considering that patients with chronic Chagas disease dis-play elevated levels of ET-1 in the bloodstream (Salomone et al., 2001), it isalso possible that trypomastigotes may induce the diffusion of bloodborne ET-1, along with kininogens and other plasma proteins (Fig. 5.1),following the sequential activation of TLR2/CXCR2>BKR/ETRs(Andrade et al., 2011). Admittedly, however, rather than exclusivelyserving as an ubiquitous gateway for parasite invasion of cardiovascularcells, BK1R engagement may also stimulate host defense by driving endo-thelium trans-migration of immunoprotective type-1 effector T cells intothe parasitized heart (Fig. 5.1). Ongoing studies should clarify if the BK1Rengagement may reciprocally intensify ETR signaling, thus forging afeedback loop that might further aggravate myocardial fibrosis duringthe chronic stage of infection.

The discovery that tGPI and cruzipain act cooperatively to activate thekinin system via the TLR2/CXCR2/neutrophil-dependent pathway(Monteiro et al., 2006) offered a paradigm to investigate the molecularbasis of the variable proinflammatory phenotypes of T. cruzi strains.Accordingly, parasite strains expressing low levels of TLR2 may not beable to efficiently induce the diffusion of plasma proteins (includingkininogens) in peripheral sites of infection. If true, we may predict thatthese parasite strains may not be capable of generating high-levels ofkinins in peripheral sites of infection, irrespective of the expression levelsof cruzipain (kinin-releasing protease). It is also possible that the proin-flammatory phenotypes of T. cruzi isolates may vary due to differences inthe efficiency of shedding of lipid vesicles bearing tGPI-linked mucins(Trocoli-Torrecilhas et al., 2009). Considering that T. cruzi is able to

perhaps favoring activation of the kinin system in TLR2-independentmanner. Another mechanism that may underlie the variable phenotype

to invade host cells expressing BKRs (influence on tissue tropism) as wellon its capacity to induce interstitial edema and T 1 responses via the

120 Julio Scharfstein and Daniele AndradeH

kinin pathway. For similar reasons, variations in the expression levels ofchagasin, a tight-binding endogenous inhibitor of papain-like cysteineproteases- originally described in T. cruzi (Monteiro et al., 2001), mayalso influence the phenotype of T. cruzi strains. This possibility is sup-ported by evidences (Aparicio et al., 2004) indicating that TCTs of the Gstrain, which are poorly infective, display increased chagasin/cruzipainratios as compared to Dm28c. Importantly, the infectivity of the G strainwas enhanced upon addition of cruzipain-rich culture supernatants fromDm28 TCTs. In the same study, the authors pointed out that that vesiclesshed by TCTsmight serve as cruzipain substrates, presumably generatinghitherto uncharacterized infection-promoting signals (Scharfstein, Lima,2008). Hence, strain-dependent differences in the expression levels of tGPIand cruzipain isoforms may influence host/parasite balance because,these factors act cooperatively, enhancing parasite infectivity while at thesame time integrating innate immunity to the proinflammatory proteo-lytic cascades that upregulate generation of TH1-type effector cells.

ACKNOWLEDGEMENTS

This research was supported by funds from the Instituto Nacional de Biologia Estruturale Bio-Imagem do CNPq; PRONEX (26/110.562/2010), FAPERJ; CNPq; financed in part byNIH Grant AI-076248 (HBT). D. A. was supported in part by a Fogarty International CenterNIH Training Grant (D43-TW007129). The authors acknowledge the help of Rafaela Serra inthe preparation of the illustration (Fig. 5.1).

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Infection-Associated Vasculopathy in Experimental Chagas Disease: Pathogenic Roles of Endothelin and Kinin PathwayIntroductionA Brief Overview on the Immunopathogenesis of Chagas DiseaseMechanisms underlying infection-associated vasculopathyBradykinin receptors: A gate of entry for Trypanosoma cruzi invasion of cardiovascular cellsInterstitial oedema induced by trypomastigotes: Role of the kinin systemACE is a negative modulator of TH1 induction by kinin danger signals released in peripheral sites of infection...DCs activated by kinins induce immunoprotective type-1 effector T cells in mice systemically infected by Trypanosoma cr

Future DirectionsAcknowledgementsReferences